The virus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) can cause persistent infection and malignant cell growth in humans. KSHV encodes two proteins, MIR1 and MIR2, that downregulate MHC-I on infected cells, .thus potentially exposing these cells to Natural Killer (NK) cell mediated lysis. We hypothesized that KSHV has also evolved a mechanism to evade killing by NK cells. Preliminary data show that one NK activating ligand, NKp44-L, is downregulated during KSHV infection, and screening for a viral gene product responsible for this activity identified the protein Kaposin B. The long-term objective of this proposal is to characterize this novel immune evasion mechanism by KSHV, and to determine whether Kaposin B allows KSHV-infected cells to evade lysis by NK cells. In Specific Aim 1, the mechanism of Kaposin B-mediated downregulation of NKp44-L will be examined. We will perform immunofluorescence and intracellular flow cytometric staining of NKp44-L, in order to determine whether ligand protein levels are decreased, and whether the protein is mislocalized, in KSHV-infected and Kaposin B-expressing cells. For Specific Aim 2, in order to determine whether Kaposin B is the activity in KSHV that causes decreased NKp44-L surface expression, Kaposin B mRNA will be knocked down during infection using siRNA. Cells will then be stained for NKp44-L to look for rescue of the downregulation. In Specific Aim 3, we will test the functional significance of NKp44-L downregulation by Kaposin B by performing in vitro NK activation and killing assays. Target cells expressing Kaposin B or a control will be co-cultured with two NKL cell lines, with and without NKp44 expression, to determine whether Kaposin B desensitizes target cells to NK killing in an NKp44-dependent manner. These experiments will also be performed using primary human NK cells, with and without blocking antibodies against NKp44. Relevance to public health: KSHV is a causative agent of several human diseases associated with immunocompromised individuals, including two B-cell lymphomas and Kaposi's Sarcoma (KS), the most common malignancy in untreated AIDS patients. NK cells are lymphocytes that are particularly important in the innate anti-viral immune response. Understanding the effects on NK cell immunity by KSHV is important for the treatment and prevention of KSHV-related diseases, and may potentially extend to related herpesviruses or other cancer- causing viruses.